Id-1 and Id-2 are overexpressed in pancreatic cancer and in dysplastic lesions in chronic pancreatitis

Abstract

Id proteins antagonize basic helix-loop-helix proteins, inhibit differentiation, and enhance cell proliferation. In this study we compared the expression of Id-1, Id-2, and Id-3 in the normal pancreas, in pancreatic cancer, and in chronic pancreatitis (CP). Northern blot analysis demonstrated that all three Id mRNA species were expressed at high levels in pancreatic cancer samples by comparison with normal or CP samples. Pancreatic cancer cell lines frequently coexpressed all three Ids, exhibiting a good correlation between Id mRNA and protein levels, as determined by immunoblotting with highly specific anti-Id antibodies. Immunohistochemistry using these antibodies demonstrated the presence of faint Id-1 and Id-2 immunostaining in pancreatic ductal cells in the normal pancreas, whereas Id-3 immunoreactivity ranged from weak to strong. In the cancer tissues, many of the cancer cells exhibited abundant Id-1, Id-2, and Id-3 immunoreactivity. Scoring on the basis of percentage of positive cells and intensity of immunostaining indicated that Id-1 and Id-2 were increased significantly in the cancer cells by comparison with the respective controls. Mild to moderate Id immunoreactivity was also seen in the ductal cells in the CP-like areas adjacent to these cells and in the ductal cells of small and interlobular ducts in CP. In contrast, in dysplastic and atypical papillary ducts in CP, Id-1 and Id-2 immunoreactivity was as significantly elevated as in the cancer cells. These findings suggest that increased Id expression may be associated with enhanced proliferative potential of pancreatic cancer cells and of proliferating or dysplastic ductal cells in CP.

Figure 1.

mRNA expression of Id-1, Id-2, and Id-3 in pancreatic cancer and chronic pancreatitis. Total RNA (20 μg/lane) from six normal, eight cancerous, and seven chronic pancreatitis tissue samples were subjected to Northern blot analysis using ³²P-labeled cDNA probes (500,000 cpm/ml) specific for Id-1, Id-2, and Id-3, respectively. A 7S cDNA probe (50,000 cpm/ml) was used as a loading and transfer control. Exposure times of the normal/cancer blots were 1 day for all Id probes, and 2 days for the normal/CP blots. Exposure time was 4 hours for mouse 7S cDNA. By comparison with the normal samples, Id-1 and Id-3 mRNA levels were elevated in 8 and 9 cancer samples, respectively, whereas Id-2 was elevated in 6 cancer samples.

Figure 2.

Densitometric analysis of Northern blots. Autoradiographs of Northern blots from 12 normal, 14 CP, and 16 pancreatic cancers were analyzed by densitometry. mRNA levels were determined by calculating the ratio of the optical density for the respective Id mRNA species in relation to the optical density of mouse 7S cDNA. To compare the relative increase in expression of the respective Id mRNA species in the cancer and CP samples, the same normal samples were used for normal/cancer and normal/CP membranes. Normal pancreatic tissues are indicated by circles, CP tissues by triangles, and cancer tissues by squares. Data are expressed as median scores ± SD. By comparison with the normal samples, only the cancer samples exhibited significant increases: 6.5-fold (P < 0.01) for Id-1, fivefold (P < 0.01) for Id-2, and twofold (P = 0.027) for Id-3.

Figure 3.

Id mRNA and protein expression in pancreatic cancer cell lines. Upper panels: Total RNA (20 μg/lane) from 5 pancreatic cancer cell lines were subjected to Northern blot analysis using ³²P-labeled cDNA probes (500,000 cpm/ml) specific for Id-1, Id-2, and Id-3, respectively. Exposure times were 1 day for all Id probes. Lower panels: Immunoblotting. Cell lysates (30 μg/lane) were subjected to SDS-PAGE. Membranes were probed with specific Id-1, Id-2, and Id-3 antibodies. Visualization was performed by enhanced chemiluminescence.

Figure 4.

Normal and cancerous pancreatic tissues were subjected to immunostaining using highly specific anti-Id-1 (A-C), anti-Id-2 (D-F), and anti-Id-3 (G-I) antibodies as described in the Methods section. Moderate to strong Id-1 immunoreactivity was present in the cytoplasm of duct-like cancer cells (A and C, left panel). In the normal pancreas there was weak Id-1 immunoreactivity in the ductal cells (B). Preabsorption with the Id-1-specific blocking peptide abolished the Id-1 immunoreactivity (C, right panel). Strong Id-2 immunoreactivity was observed in the cytoplasm of the cancer cells that exhibited duct-like structures (D and F, left panel), whereas in the normal pancreas, there was only weak Id-2 immunoreactivity in the ductal cells (E). Preabsorption with the Id-2-specific blocking peptide abolished the Id-2 immunoreactivity (F, right panel). Moderate to strong Id-3 immunoreactivity was present in the duct-like cancer cells (G and I, left panel). Moderate to strong Id-3 immunoreactivity was also present in the ductal cells of normal pancreatic tissue samples (H). Id-3 immunoreactivity was completely abolished by preabsorption with the Id-3 specific blocking peptide (I, right panel) . A, D, and G constitute serial sections of a pancreatic cancer sample, revealing coexpression of the three Id proteins. Scale bars, 25 μm.

Figure 5.

Immunohistochemistry of pancreatic cancer and dysplastic ducts in CP tissues. In the pancreatic cancer tissues (A-C) there was moderate to strong Id-1 (A), Id-2 (B), and Id-3 (C) immunoreactivity in the ductal cells in the areas adjacent to the cancer cells that exhibited CP-like alterations. Islet cells did not exhibit Id immunoreactivity (outlined by solid arrowheads). In the CP samples, moderate to strong Id-1 (D), Id-2 (E), and Id-3 (F) immunoreactivity was present in the cytoplasm of epithelial cells forming large dysplastic ducts. Scale bar, 25 μm.

Figure 6.

Immunohistochemistry of atypical papillary epithelium in CP tissues. Serial section analysis of some CP samples revealed the presence of large duct-like structures with atypical papillary epithelium. Mild to moderate Id-1 (A) and Id-2 (B) immunoreactivity and weak Id-3 (C) immunoreactivity was present in the cytoplasm of the cells forming these large ducts with papillary structures. Some CP samples also exhibited moderate Id-3 immunoreactivity in these cells (D). Scale bar, 25 μm.